Structural basis of Integrator-dependent RNA polymerase II termination.

The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.

Structural characterization of the ANTAR antiterminator domain bound to RNA.

Regulated transcription termination provides an efficient and responsive means to control gene expression. In bacteria, rho-independent termination occurs through the formation of an intrinsic RNA terminator loop, which disrupts the RNA polymerase elongation complex, resulting in its dissociation from the DNA template. Bacteria have a number of pathways for overriding termination, one of which is the formation of mutually exclusive RNA motifs. ANTAR domains are a class of antiterminator that bind and stabilize dual hexaloop RNA motifs within the nascent RNA chain to prevent terminator loop formation. We have determined the structures of the dimeric ANTAR domain protein EutV, from Enterococcus faecialis, in the absence of and in complex with the dual hexaloop RNA target. The structures illustrate conformational changes that occur upon RNA binding and reveal that the molecular interactions between the ANTAR domains and RNA are restricted to a single hexaloop of the motif. An ANTAR domain dimer must contact each hexaloop of the dual hexaloop motif individually to prevent termination in eubacteria. Our findings thereby redefine the minimal ANTAR domain binding motif to a single hexaloop and revise the current model for ANTAR-mediated antitermination. These insights will inform and facilitate the discovery of novel ANTAR domain RNA targets.

Cyclic peptides can engage a single binding pocket through highly divergent modes.

Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and ß-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.

BET-Family Bromodomains Can Recognize Diacetylated Sequences from Transcription Factors Using a Conserved Mechanism.

Almost all eukaryotic proteins receive diverse post-translational modifications (PTMs) that modulate protein activity. Many histone PTMs are well characterized, heavily influence gene regulation, and are often predictors of distinct transcriptional programs. Although our understanding of the histone PTM network has matured, much is yet to be understood about the roles of transcription factor (TF) PTMs, which might well represent a similarly complex and dynamic network of functional regulation. Members of the bromodomain and extra-terminal domain (BET) family of proteins recognize acetyllysine residues and relay the signals encoded by these modifications. Here, we have investigated the acetylation dependence of several functionally relevant BET-TF interactions in vitro using surface plasmon resonance, nuclear magnetic resonance, and X-ray crystallography. We show that motifs known to be acetylated in TFs E2F1 and MyoD1 can interact with all bromodomains of BRD2, BRD3, and BRD4. The interactions are dependent on diacetylation of the motifs and show a preference for the first BET bromodomain. Structural mapping of the interactions confirms a conserved mode of binding for the two TFs to the acetyllysine binding pocket of the BET bromodomains, mimicking that of other already established functionally important histone- and TF-BET interactions. We also examined a motif from the TF RelA that is known to be acetylated but were unable to observe any interaction, regardless of the acetylation state of the sequence. Our findings overall advance our understanding of BET-TF interactions and suggest a physical link between the important diacetylated motifs found in E2F1 and MyoD1 and the BET-family proteins.

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae.

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.

The molecular dynamics of long noncoding RNA control of transcription in PTEN and its pseudogene.

RNA has been found to interact with chromatin and modulate gene transcription. In human cells, little is known about how long noncoding RNAs (lncRNAs) interact with target loci in the context of chromatin. We find here, using the phosphatase and tensin homolog (PTEN) pseudogene as a model system, that antisense lncRNAs interact first with a 5' UTR-containing promoter-spanning transcript, which is then followed by the recruitment of DNA methyltransferase 3a (DNMT3a), ultimately resulting in the transcriptional and epigenetic control of gene expression. Moreover, we find that the lncRNA and promoter-spanning transcript interaction are based on a combination of structural and sequence components of the antisense lncRNA. These observations suggest, on the basis of this one example, that evolutionary pressures may be placed on RNA structure more so than sequence conservation. Collectively, the observations presented here suggest a much more complex and vibrant RNA regulatory world may be operative in the regulation of gene expression.

Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5.

Changes within the central stalk of E. coli F1Fo ATP synthase observed after addition of ATP.

F1Fo ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the Fo motor that is transferred to the F1 motor to catalyze ATP production, with flexible F1/Fo coupling required for efficient catalysis. F1Fo ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory ε subunit. Here we present cryo-EM data showing E. coli F1Fo ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory ε subunit induce torsional movement in the central stalk, thereby enabling its rotation within the Fο motor. This highlights the importance of the central rotor for flexible coupling of the F1 and Fo motors and provides further insight into the regulatory mechanism mediated by subunit ε.

Driving forces behind phase separation of the carboxy-terminal domain of RNA polymerase II.

Eukaryotic gene regulation and pre-mRNA transcription depend on the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II. Due to its highly repetitive, intrinsically disordered sequence, the CTD enables clustering and phase separation of Pol II. The molecular interactions that drive CTD phase separation and Pol II clustering are unclear. Here, we show that multivalent interactions involving tyrosine impart temperature- and concentration-dependent self-coacervation of the CTD. NMR spectroscopy, molecular ensemble calculations and all-atom molecular dynamics simulations demonstrate the presence of diverse tyrosine-engaging interactions, including tyrosine-proline contacts, in condensed states of human CTD and other low-complexity proteins. We further show that the network of multivalent interactions involving tyrosine is responsible for the co-recruitment of the human Mediator complex and CTD during phase separation. Our work advances the understanding of the driving forces of CTD phase separation and thus provides the basis to better understand CTD-mediated Pol II clustering in eukaryotic gene transcription.

The bromodomains of BET family proteins can recognize diacetylated histone H2A.Z.

Chemical modifications of histone tails influence genome accessibility and the transcriptional state of eukaryotic cells. Lysine acetylation is one of the most common modifications and acetyllysine-binding bromodomains (BDs) provide a means for acetyllysine marks to be translated into meaningful cellular responses. Here, we have investigated the mechanism underlying the reported association between the Bromodomain and Extra Terminal (BET) family of BD proteins and the essential histone variant H2A.Z. We use NMR spectroscopy to demonstrate a physical interaction between the N-terminal tail of H2A.Z and the BDs of BRD2, BRD3, and BRD4, and show that the interaction is dependent on lysine acetylation in H2A.Z. The BDs preferentially engage a diacetylated H2A.Z-K4acK7ac motif that is reminiscent of sequences found in other biologically important BET BD target proteins, including histones and transcription factors. A H2A.Z-K7acK11ac motif can also bind BET BDs-with a preference for the second BD of each protein. Chemical shift perturbation mapping of the interactions, together with an X-ray crystal structure of BRD2-BD1 bound to H2A.Z-K4acK7ac, shows that H2A.Z binds the canonical AcK binding pocket of the BDs. This mechanism mirrors the conserved binding mode that is unique to the BET BDs, in which two acetylation marks are read simultaneously by a single BD. Our findings provide structural corroboration of biochemical and cell biological data that link H2A.Z and BET-family proteins, suggesting that the function of H2A.Z is enacted through interactions with these chromatin readers.

Perceptions and attitudes toward risk and personal responsibility in the context of medical fitness to drive.

Objective: Although there is a considerable body of literature probing the knowledge and awareness of doctors of guidelines on medical fitness to drive, little is known of knowledge and awareness of such guidelines among the general public. We investigated awareness, perceptions and attitudes among a range of adults toward risk and personal responsibility in the context of medical fitness to drive.Methods: This quasi-experimental between groups study assessed response differences between healthcare (33%) and non-healthcare professionals. Five hundred and fourteen adults (f = 342) completed a 15-item online survey, which assessed their license status, direct and secondhand experience with key medical conditions, perceptions of risk, driving intentions if advised to stop driving by a doctor and beliefs about responsibility for ensuring driver fitness.Results: Most of the participants had a driver license. Although the majority (87%) had no first-hand experience with key medical conditions contained in the Irish medical fitness to drive guidelines, two-thirds knew someone with at least one of these conditions. No participant admitted to an alcohol or drug misuse/dependence problem. Alcohol misuse/dependence and fatigue/chronic sleep loss were perceived as the greatest crash risk for drivers and for vulnerable road users. Risk perceptions in general public and the medical professionals were similar for most conditions but where they differed, the medical professionals perceived lower risk. Most respondents indicated that they would cease driving immediately if advised by their doctor for temporary and long-term conditions. No effects of age, gender, experience with medical conditions or professional status were noted on perceived intentions to cease driving. Perceptions about who is responsible for ensuring people are fit to drive fell into two categories. Drivers were perceived as chiefly responsible where alcohol, non-prescription drugs, fatigue and age were factors and doctors were identified as responsible in cases of diabetes, epilepsy, stroke, heart conditions and physical disabilities.Conclusions: These results suggest that more needs to be done to raise awareness of the risks posed by medical and other factors that reduce driver capability and also about drivers' responsibility to ensure that they are fit to drive and thus improve road safety.

Nanofluid Development Using Silver Nanoparticles and Organic-Luminescent Molecules for Solar-Thermal and Hybrid Photovoltaic-Thermal Applications.

Exploiting solar energy using photo-thermal (PT) and/or hybridised photovoltaic/thermal (PVT) systems can represent a viable alternative to the growing demand for renewable energy. For large-scale implementation, such systems require thermal fluids able to enhance the combined conversion efficiency achievable by controlling the 'thermal' and 'electrical' components of the solar spectrum. Nanofluids are typically employed for these purposes and they should exhibit high heat-transfer capabilities and optical properties tuned towards the peak performance spectral window of the photovoltaic (PV) component. In this work, novel nanofluids, composed of highly luminescent organic molecules and Ag nanoparticles dispersed within a base fluid, were tested for PT and PVT applications. These nanofluids were designed to mimic the behaviour of luminescent down-shifting molecules while offering enhanced thermo-physical characteristics over the host base fluid. The nanofluids' conversion efficiency was evaluated under a standard AM1.5G weighted solar spectrum. The results revealed that the Ag nanoparticles' inclusion in the composite fluid has the potential to improve the total solar energy conversion. The nanoparticles' presence minimizes the losses in the electrical power component of the PVT systems as the thermal conversion increases. The enhanced performances recorded suggest that these nanofluids could represent suitable candidates for solar energy conversion applications.

Cryo-EM structures provide insight into how E. coli F1Fo ATP synthase accommodates symmetry mismatch.

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. It comprises two molecular motors coupled together by a central and a peripheral stalk. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production. Here we present nine cryo-EM structures of E. coli ATP synthase to 3.1-3.4 Å resolution, in four discrete rotational sub-states, which provide a comprehensive structural model for this widely studied bacterial molecular machine. We observe torsional flexing of the entire complex and a rotational sub-step of Fo associated with long-range conformational changes that indicates how this flexibility accommodates the mismatch between the 3- and 10-fold symmetries of the F1 and Fo motors. We also identify density likely corresponding to lipid molecules that may contribute to the rotor/stator interaction within the Fo motor.

Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana.

Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel retardation assays and microscale thermophoresis with two different oligonucleotide probes containing one and two copies of recognition sequence AAAG. Results: DOF zinc finger domains (DOF-ZFs) were shown to form independently folded structures. Assessments using microscale thermophoresis demonstrated that DOF-ZFs interact more tightly (~ 100 fold) with double-motif probe than the single-motif probe. The overall Kd values for the DOF3.4-ZF and DOF5.8-ZF to the double-motif probe were ~2.3±1 and 2.5±1 µM, respectively. Conclusion: Studied DOF-ZF domains formed stable complexes with the double-motif probe. Although DOF3.4-ZF and DOF5.8-ZF do not dimerize with an appreciable affinity in the absence of DNA (judging from size-exclusion and multiangle laser light scattering data), it is possible that these ZFs form protein-protein contacts when bound to this oligonucleotide, consistent with previous reports that DOF proteins can homo- and hetero-dimerize.

The structure of the neisserial lipooligosaccharide phosphoethanolamine transferase A (LptA) required for resistance to polymyxin.

Gram-negative bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a soluble catalytic domain. The crystal structure of the soluble domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been determined crystallographically and refined to 1.4Å resolution. The structure reveals a core hydrolase fold similar to that of alkaline phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is observed. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chemistry; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.